The overall objective of this plan will be to facilitate the development of the candidate into a highly trained scientist in the area of ischemic preconditioning (PC) of skeletal muscle to improve dynamic cardiomyoplasty. Ischemic PC is the phenomenon whereby brief ischemic episodes render skeletal muscle resistant to subsequent ischemia. Two windows of protection have been described: an early phase which occurs immediately after PC, and a delayed phase which manifests itself 24 hours after PC. The applicants hypothesize that the delayed phase of ischemic PC in skeletal muscle is mediated by activation of one or few individual PKC isoform(s). The specific aims are: 1) optimize the PC protocol for the delayed phase of protection; 2) determine the duration of the delayed phase of protection; 3) to fully characterize the activation of individual PKC isoforms during ischemic and pharmacological PC; 4) to determine the effect of PKC inhibitors on individual PKC isoform translocation; 5) to define in isolated myocytes the role of PKC isoform(s) responsible for the delayed phase of protection; and 6) to construct a transgenic mouse that overexpresses PKC isoforms responsible for PC. Rat latissimus dorsi muscle (LDM) flaps will be used in 4 studies. 1) LDMs will be preconditioned with ischemia and with monophosphoryl lipid A (MLA). Twenty-four hours after PC, muscles will be subjected to 4 h of ischemia, and 72 h later infarct size will be quantified. 2) LDMs will be PC with ischemia and MLA 4) Chelerythrine will be given prior to PC and biopsies taken after ischemic PC and MLA. In in vitro studies, using a model of anoxia-reoxygenation to mimic PC, myocytes will be preconditioned and 24 h later submitted to anoxia, and cell death will be the end-point. The last study will involve the construct of Transgenic mice overexpressing PKC isoforms responsible for PC. The findings of this project will provide new information regarding the mechanisms of ischemic PC and important knowledge regarding the pathogenesis of ischemia in skeletal muscle. (End of Abstract)